Biology subjects

Deutschbauer, A.

Publications and source records attributed to Deutschbauer, A..

27 records · Page 2Linked to original sources

bioRxiv2021

Construction and characterization of a genome-scale ordered mutant collection of Bacteroides thetaiotaomicron

Genomic analyses have revealed how the gut microbiota impacts human health. However, knowledge about the physiology of most gut commensals is largely lacking. Here, we sorted cells from a pooled library to construct an ordered collection of transposon-insertion mutants in the model commensal Bacteroides thetaiotaomicron. We applied a pooling strategy with barcode sequencing to locate mutants and created a condensed collection with single insertions in 2,565 genes. This effort enabled the development of an accurate model for progenitor-collection assembly, which identified strain-abundance biases and multi-insertion strains as important factors that limit coverage. To demonstrate the potential for phenotypic screening, we analyzed growth dynamics and morphology of the condensed collection and identified growth defects and altered cell shape in the sphingolipid-synthesis gene BT0870 and the thiamine scavenging gene BT2397. Analyses of this collection and utilization of the platform described herein to construct future ordered libraries will increase understanding of gut commensal physiology and colonization strategies.

microbiology↗

bioRxiv2021

Systematic dissection of a complex gut bacterial community

Efforts to model the gut microbiome have yielded important insights into the mechanisms of interspecies interactions, the impact of priority effects on ecosystem dynamics, and the role of diet and nutrient availability in determining community composition. However, the model communities studied to date have been defined or complex but not both, limiting their utility. Here, we construct a defined community of 104 bacterial strains composed of the most common taxa from the human gut microbiota. By propagating this community in growth media missing one amino acid at a time, we show that branched-chain amino acids have an outsize impact on community structure and identify a pathway in Clostridium sporogenes for generating ATP from arginine. We constructed and propagated the complete set of single-strain dropout communities, revealing a sparse network of strain-strain interactions including a novel interaction between C. sporogenes and Lactococcus lactis driven by metabolism. This work forms a foundation for studying strain-strain and strain-nutrient interactions in highly complex defined communities, and it provides a starting point for interrogating the rules of synthetic ecology at the 100+ strain scale.

microbiology↗

bioRxiv2021

High Throughput Fitness Profiling Reveals Loss Of GacS-GacA Regulation Improves Indigoidine Production In Pseudomonas putida

Pseudomonas putida KT2440 is an emerging industrial microbe amenable for use with renewable carbon streams including aromatics such as para-coumarate (pCA). We examined this microbe under common stirred-tank bioreactor parameters with quantitative fitness assays using a pooled transposon library containing nearly all (4,778) non-essential genes. Assessing differential fitness values by monitoring changes in mutant strain abundance over time identified 31 genes with improved fitness in multiple bioreactor-relevant parameters. Twenty-one genes from this subset were reconstructed, including GacA, a signaling protein, TtgB, an ABC transporter, and PP_0063, a lipid A acyltransferase. Twelve deletion strains with roles in varying cellular functions were evaluated for conversion of pCA, to a heterologous bioproduct, indigoidine. Several mutants, such as the {Delta}gacA strain improved both fitness in a bioreactor and showed an 8-fold improvement in indigoidine production (4.5 g/L, 0.29 g/g pCA, 23% MTY) from pCA as the carbon source.

systems biology↗

bioRxiv2020

Distinctiveness of genes contributing to growth of Pseudomonas syringae in diverse host plant species

A variety of traits are necessary for bacterial colonization of the interior of plant hosts, including well-studied virulence effectors as well as other phenotypes contributing to bacterial growth and survival within the apoplast. High-throughput methods such as transposon sequencing (TnSeq) are powerful tools to identify such genes in bacterial pathogens. However, there is little information as to the distinctiveness of traits required for bacterial colonization of different hosts. Here, we utilize randomly barcoded TnSeq (RB-TnSeq) to identify the genes that contribute to the ability of Pseudomonas syringae strain B728a to grow within common bean (Phaseolus vulgaris), lima bean (Phaseolus lunatus), and pepper (Capsicum annuum); species representing two different plant families. The magnitude of contribution of most genes to apoplastic fitness in each of the plant hosts was similar. However, 50 genes significantly differed in their fitness contributions to growth within these species. These genes encoded proteins in various functional categories including polysaccharide synthesis and transport, amino acid metabolism and transport, cofactor metabolism, and phytotoxin synthesis and transport. Six genes that encoded unannotated, hypothetical proteins also contributed differentially to growth in these hosts. The genetic repertoire of a relatively promiscuous pathogen such as P. syringae may thus be shaped, at least in part, by the conditional contribution of some fitness determinants.

microbiology↗

bioRxiv2020

Functional analysis of the fatty acid and alcohol metabolism of Pseudomonas putida using RB-TnSeq

ABSTRACTWith its ability to catabolize a wide variety of carbon sources and a growing engineering toolkit, Pseudomonas putida KT2440 is emerging as an important chassis organism for metabolic engineering. Despite advances in our understanding of this organism, many gaps remain in our knowledge of the genetic basis of its metabolic capabilities. These gaps are particularly noticeable in our understanding of both fatty acid and alcohol catabolism, where many paralogs putatively coding for similar enzymes co-exist making biochemical assignment via sequence homology difficult. To rapidly assign function to the enzymes responsible for these metabolisms, we leveraged Random Barcode Transposon Sequencing (RB-TnSeq). Global fitness analyses of transposon libraries grown on 13 fatty acids and 10 alcohols produced strong phenotypes for hundreds of genes. Fitness data from mutant pools grown on varying chain length fatty acids indicated specific enzyme substrate preferences, and enabled us to hypothesize that DUF1302/DUF1329 family proteins potentially function as esterases. From the data we also postulate catabolic routes for the two biogasoline molecules isoprenol and isopentanol, which are catabolized via leucine metabolism after initial oxidation and activation with CoA. Because fatty acids and alcohols may serve as both feedstocks or final products of metabolic engineering efforts, the fitness data presented here will help guide future genomic modifications towards higher titers, rates, and yields.IMPORTANCE To engineer novel metabolic pathways into P. putida, a comprehensive understanding of the genetic basis of its versatile metabolism is essential. Here we provide functional evidence for the putative roles of hundreds of genes involved in the fatty acid and alcohol metabolism of this bacterium. These data provide a framework facilitating precise genetic changes to prevent product degradation and channel the flux of specific pathway intermediates as desired.Competing Interest StatementJ.D.K. has financial interests in Amyris, Lygos, Demetrix, Napigen, Maple Bio, and Apertor Labs. C.B.E has a financial interest in Perlumi Chemicals.View Full Text

microbiology↗

bioRxiv2020

Iron Supplementation Eliminates Antagonistic Interactions Between Root Associated Bacteria

The rhizosphere microbiome (rhizobiome) plays a critical role in plant health and development. However the processes by which the constituent microbes interact to form and maintain a community are not well understood. To investigate these molecular processes, we examined pairwise interactions between 11 different microbial isolates under selected nutrient-rich and nutrient-limited conditions. We observed that when grown with media supplemented with 56 mM glucose, 2 microbial isolates were able to inhibit the growth of 6 out of 11 other microbes tested. The interaction between microbes persisted even after the antagonistic microbe was removed, upon exposure to spent media. To probe the genetic basis for these antagonistic interactions, we used a barcoded transposon library in a proxy bacterium, Pseudomonas putida, to identify genes which showed enhanced sensitivity to the antagonistic factor(s) secreted by Acinetobacter sp. 02. Iron metabolism-related gene clusters in P. putida were implicated by this systems-level analysis. The supplementation of iron prevented the antagonistic interaction in the original microbial pair supporting the hypothesis that iron limitation drives antagonistic microbial interactions between rhizobionts. We conclude that rhizobiome community composition is influenced by competition for limiting nutrients with implications for growth and development of the plant.

microbiology↗

bioRxiv2019

Leveraging host metabolism for bisdemethoxycurcumin production in Pseudomonas putida

Pseudomonas putida is a saprophytic bacterium with robust metabolisms and strong solvent tolerance making it an attractive host for metabolic engineering and bioremediation. Due to its diverse carbon metabolisms, its genome encodes an array of proteins and enzymes that can be readily applied to produce valuable products. In this work we sought to identify design principles and bottlenecks in the production of type III polyketide synthase (T3PKS)-derived compounds in P. putida. T3PKS products are widely used as nutraceuticals and medicines and often require aromatic starter units, such as coumaroyl-CoA, which is also an intermediate in the native coumarate catabolic pathway of P. putida. Using a randomly barcoded transposon mutant (RB-TnSeq) library, we assayed gene functions for a large portion of aromatic catabolism, confirmed known pathways, and proposed new annotations for two aromatic transporters. The 1,3,6,8-tetrahydroxynapthalene synthase of Streptomyces coelicolor (RppA), a microbial T3PKS, was then used to rapidly assay growth conditions for increased T3PKS product accumulation. The feruloyl/coumaroyl CoA synthetase (Fcs) of P. putida was used to supply coumaroyl-CoA for the curcuminoid synthase (CUS) of Oryza sativa, a plant T3PKS. We identified that accumulation of coumaroyl-CoA in this pathway results in extended growth lag times in P. putida. Deletion of the second step in coumarate catabolism, the enoyl-CoA hydratase-lyase (Ech), resulted in increased production of the type III polyketide bisdemethoxycurcumin.

microbiology↗

bioRxiv2019

Host engineering for improved valerolactam production in Pseudomonas putida

Pseudomonas putida is a promising bacterial chassis for metabolic engineering given its ability to metabolize a wide array of carbon sources, especially aromatic compounds derived from lignin. However, this omnivorous metabolism can also be a hindrance when it can naturally metabolize products produced from engineered pathways. Herein we show that P. putida is able to use valerolactam as a sole carbon source, as well as degrade caprolactam. Lactams represent important nylon precursors, and are produced in quantities exceeding one million tons per year[1]. To better understand this metabolism we use a combination of Random Barcode Transposon Sequencing (RB-TnSeq) and shotgun proteomics to identify the oplBA locus as the likely responsible amide hydrolase that initiates valerolactam catabolism. Deletion of the oplBA genes prevented P. putida from growing on valerolactam, prevented the degradation of valerolactam in rich media, and dramatically reduced caprolactam degradation under the same conditions. Deletion of oplBA, as well as pathways that compete for precursors L-lysine or 5-aminovalerate, increased the titer of valerolactam from undetectable after 48 hours of production to ~90 mg/L. This work may serve as a template to rapidly eliminate undesirable metabolism in non-model hosts in future metabolic engineering efforts.

synthetic biology↗

bioRxiv2019

Large-scale chemical-genetics of the human gut bacterium Bacteroides thetaiotaomicron

The genomic catalogue of the human microbiota has expanded dramatically in recent years, and insights derived from human microbiota genomics has vast potential to generate treatments for human diseases. However, predictably harnessing the microbiota for beneficial outcomes is currently limited by our lack of understanding of the physiology of the constituent bacteria. For instance, the functions of most of their genes are not known. Here, we systematically measure mutant phenotypes for genes from the gut commensal Bacteroides thetaiotaomicron. Using a barcoded transposon mutant library, we measured the fitness of 4,055 B. thetaiotaomicron genes across 492 experiments, including growth on 45 carbon substrates and in the presence of 57 stress-inducing compounds. Our data is in strong agreement with previous studies, and more importantly also uncovers the biological roles of poorly annotated genes. We identified 497 genes with a specific phenotype in only one or a handful of conditions, thus enabling informed predictions of gene function for a subset of these genes. For example, we identified a glycoside hydrolase important for growth on type I rhamnogalacturonan, a DUF4861 protein for glycosaminoglycan utilization, a DUF1080 protein for disaccharide utilization, and a tripartite multidrug resistance system specifically important for bile salt tolerance. Our approach can be applied to other members of the human microbiota to experimentally characterize their genes.

microbiology↗